Of Seven New K+ Channel Inhibitor Peptides of Centruroides bonito, α-KTx 2.24 Has a Picomolar Affinity for Kv1.2

Abstract

Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three pu-tative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24–763 pM) and inhibited the Kv1.3 channel with comparatively less po-tency (Kd values between 20–171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 in-hibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.

Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito

“…With this idea, Fraction II was further separated by ion-exchange chromatography on a carboxy–methyl–cellulose (CMC) column (5 mL volume) run in the same buffer and eluted with a sodium chloride gradient from 0 to 0.5 M salt, during 1000 min at 0.5 mL/min, in a New Generation Chromatography (NGC TM ) system connected to a fraction collector (both from BioRAD, Mexico City, Mexico). Fractions from the CMC column were further separated through high-performance liquid chromatography (HPLC) using a Waters model 1525 binary HPLC pump, detector 2489 UV/vis chromatographer (Mexico City, Mexico) in the conditions earlier described [ 45 ]. Basically, an analytical C18 reverse-phase column from Vydac (Hisperia, CA, USA) was used (C18 250 × 4.6 mm, wakopac/wakosil pTH–II 4.6 × 250 mm).…”

“…In our conditions, most pure peptides in their native format allow for the identification of approximately the first 50 amino acid residues, starting from the N–terminal amino acid. In order to obtain the full sequence, the reduced and alkylated format of the peptides was submitted to enzymatic hydrolysis [ 45 ]. The corresponding peptides after treatment with the endopeptidases (GluC and AspN) were subjected to HPLC separation, and their sub-peptides were placed into the sequencer.…”